Chemical product usable as a non-radioactive carrier

ABSTRACT

A radioactive diagnostic agent which comprises (1) the unit of a polyformyl compound having at least three formyl groups per molecule, (2) at least two units of an amino group-containing chelating compound bonded to the polyformyl compound with intervention of a methyleneimine linkage (--CH═N--) or a methyleneamine linkage (--CH 2  NH--) formed by the condensation between the formyl group in the polyformyl compound and the amino group in the chelating compound, optionally followed by reduction, (3) at least one unit of an amino group-containing physiologically active substance bonded to the polyformyl compound with intervention of a methyleneimine linkage or a methyleneamine linkage formed by the condensation between the formyl group in the polyformyl compound and the amino group in the physiologically active substance, optionally followed by reduction, and (4) at least two radioactive metallic elements of which each is bonded to the chelating compound through a chelating bond.

This application is a continuation of application Ser. No. 07/771,218filed on Oct. 4, 1991, now abandoned, which was a divisional of07/947,093 filed Dec. 29, 1986, now U.S. Pat. No. 5,077,389, which was adivisional of 07/558,333, filed on Dec. 5, 1983, now U.S. Pat. No.4,666,697.

The present invention relates to a radioactive diagnostic agent, andnon-radioactive carriers therefor. More particularly, it relates to aradioactive diagnostic agent which comprises a formyl group-containing,chelating substance comprising the unit of a polyformyl compound and theunit of an amino group-containing chelating compound in combination, andan amino group-containing physiologically active substance and aradioactive metallic element bonded to said chelating substance, andnon-radioactive carriers therefor.

For the purpose of a non-invading (i.e., non-surgical) nuclear medicaldiagnosis such as recording, dynamic study and quantitative measurementof the blood circulation system, detection of physiological abnormalityor localization of abnormality by imaging, there have been widely usedphysiologically active substances labeled with iodine-131 (¹³¹ I) (i.e.,¹³¹ I-labeled serum albumin and ¹³¹ I-labeled fibrinogen). However, ¹³¹I has a long half life of about 8 days and emits beta-rays so that thepatient administered therewith is exposed to a large quantity ofradiation.

In order to overcome the above-mentioned drawback in ¹³¹ I-labeledphysiologically active substances, attempts have been made to provideradioactive diagnostic agents comprising physiologically activesubstances and radioactive metallic elements having more favorablephysical properties than iodine-131. For example, there is a knownlabeling method wherein a physiologically active substance is directlytreated with a radioactive metal salt to make a chelate compound whichmay be used as a radioactive diagnostic agent. For instance, human serumalbumin is treated with an aqueous solution containing technetium-99m(^(99m) Tc) in the form of pertechnetate in the presence of a reducingagent to give ^(99m) Tc-labeled human serum albumin. In another example,bleomycin is treated with an aqueous solution containing indium-111 (¹¹¹In) in the form of indium chloride to give ¹¹¹ In-labeled bleomycin.However, the chelate forming property of those physiologically activesubstances is not sufficient and the once formed chelating bond isreadily broken. In fact, ^(99m) Tc-labeled serum albumin and ¹¹¹In-labeled bleomycin are so unstable after administration into livingbodies that the behavior of the radioactivity in such bodies does notcoincide with that of serum albumin or bleomycin of the physiologicallyactive substance. This is a fatal defect for the nuclear medicaldiagnosis based on the exact trace of the behavior of the radioactivitywhich should coincide with the behavior of the physiologically activesubstance.

In recent years, attention was drawn to some chelating compounds whichon one hand show a strong chelate forming property to a variety ofmetals and on the other hand have an amino group or a carboxyl grouphighly reactive to various physiologically active substances. Attemptshave been made to utilize these characteristic features and combine aradioactive metallic element and a physiologically active substance tothem. Examples of such chelating compounds arediethylenetriamine-pentaacetic acid, ethylenediamine-triacetic acid,3-oxobutyral-bis(N-methylthiosemicarbazone)carboxylic acid,deferoxamine, 3-aminomethylene-2,4-pentanedione-bis(thiosemicarbazone)derivatives, 1- (p-aminoalkyl)phenylpropane-1,2-dione-bis(N-methylthiosemicarbazone) derivatives, etc.[Krejcarek: Biochemical & Biophysical Research Comm, Vol. 77, 2,581-585(1977); Leurg: Int. J. Appl. Radiation & Isotopes, Vol. 29, 687-692(1978); Japanese Patent Publn. (unexamined) Nos. 56-34634, 56-125317,57-102820, etc.]. Since the resulting products are stable and retain theactivities of the physiologically active substances combined therein,they are suitable for the diagnostic use. However, the products combinedwith the physiologically active substances which usually have a largemolecular weight such as fibrinogen (molecular weight, about 340,000)and IgG (molecular weight, about 160,000) can hardly provide asufficiently high radioactivity as necessitated for diagnosis.

In order to overcome the above-mentioned drawback, a combinationcontaining a physiologically active substance having many chelatingcompounds may be combined with many radioactive metallic elements. Whilethis method will assure high radioactivity, the resultingphysiologically active substance may be unfavorably denatured or itsphysiological activity may be undesirably reduced or lost.

Furthermore, a physiologically active substance having a large molecularweight is preferably administered to human beings at smaller doses inview of its antigen property. For realization of such administration,the physiologically active substance is also preferred to have a higherradioactivity.

As a result of extensive study, it has now been found that the use of aformyl group-containing, chelating substance comprising a unit of apolyformyl compound and a unit of an amino group-containing chelatingcompound in combination as a carrier for a physiologically activesubstance and a radioactive metallic element forms a radioactivediagnostic agent. This agent has a relatively high radioactivity permolecule without causing any deterioration or decrease in thephysiological activity inherent to the physiologically active substance.

According to the present invention, a radioactive diagnostic agent whichcomprises (1) a unit of a polyformyl compound having at least threeformyl groups per molecule, (2) at least two units of an amino groupcontaining a chelating compound bonded to the polyformyl compound by ofa methylenimine linkage (--CH═N--) or a methylenamine linkage (--CH₂NH--) formed by the condensation between the formyl group in thepolyformyl compound and the amino group in the chelating compound,optionally followed by reduction, (3) at least one unit of an aminogroup-containing a physiologically active substance bonded to thepolyformyl compound by a methylenimine linkage or a methylenaminelinkage formed by the condensation between the formyl group in thepolyformyl compound and the amino group in the physiologically activesubstance, optionally followed by reduction, and (4) at least tworadioactive metallic elements of which each is bonded to the chelatingcompound through a chelating bond.

A non-radioactive carrier is also provided which comprises a unit of apolyformyl compound having at least three formyl groups, and (2) atleast two units of an amino group-containing chelating compound bondedto the polyformyl compound by a methylenimine linkage or a methylenaminelinkage which is formed by the condensation between the formyl group inthe polyformyl compound and the amino group in the chelating compound,optionally followed by reduction, and being useful for preparation ofsaid radioactive diagnostic agent.

A physiologically active substance which is combined with anon-radioactive carrier is further provided which comprises (1) unit ofa polyformyl compound having at least three formyl groups, (2) at leasttwo units of an amino group-containing chelating compound which isbonded to the polyformyl compound by a methyleneimine linkage or amethylenenamine linkage formed by the condensation between the formylgroup in the polyformyl compound and the amino group in the chelatingcompound, optionally followed by reduction, and (3) at least one unit ofan amino group-containing a physiologically active substance bonded tothe polyformyl compound by a methylenimine linkage or a methylenaminelinkage which is formed by the condensation between the formyl group inthe polyformyl compound and the amino group in the physiologicallyactive substance, optionally followed by reduction, and being useful forpreparation of said radioactive diagnostic agent.

The polyformyl compound (1) is required to have at least three formylgroups in the molecule and preferably has a higher number of formylgroups. Among those formyl groups, at least two are combined with acorresponding number on the amino group-containing chelating compound(2), and at least one is combined with a corresponding number on thephysiologically active substance (3). Specific examples of thepolyformyl compound (1) include polyacrolein, polyethacrolein, etc. Thepreferred compound is polyacrolein of the formula: ##STR1## wherein p isusually from 3 to 4000 and preferably from 10 to 500. The polyacroleinmay be prepared, for instance, by subjecting acrolein to Redoxpolymerization [Schulz et al.: Makromol. Chem. , Vol. 24, page 141(1957)]. Other specific examples include poly(dialdehydosaccharides),and a typical one is a dialdehydo-starch of the formula: ##STR2##wherein p' is usually from 2 to 1000, preferably from 10 to 500. Thesemay be prepared, for instance, by oxidizing polysaccharides (e.g.starch, amylose, dextran, purdan) with an oxidizing agent (e.g. sodiumperiodate) so as to form two formyl groups in each saccharide unit.

The amino group-containing chelating compound (2), may be any suchcompound showing a strong chelate forming property to a radioactivemetallic element and has an amino group capable of reacting to a formylgroup in the polyformyl compound (1) under a relatively mild condition.Specific examples are deferoxamine (i.e.1-amino-6,17-di-hydroxy-7,10,18,21-tetraoxo-27-(N-acetyl-hydroxylamino)-6,11,17,22-tetraazaheptaeicosane)[The Merck Index, 9th Ed., page 374 (1976)],3-aminomethylene-2,4-pentanedione-bis(thiosemicarbazone) derivatives ofthe formula: ##STR3## wherein R¹ and R² are each a hydrogen atom, a C₁-C₃ alkyl group or a phenyl group [EP-A-0054920], 1-(p-aminoalkyl)phenylpropane-1,2-dione-bis(thiosemicarbazone) derivatives of theformula: ##STR4## wherein R³ and R⁴ are each a hydrogen atom or a C₁ -C₃alkyl group and n is 0 or an integer of 1 to 3 [Australian patent533722], etc. Any compound which has a metal capturing property to forma chelate and does not have an amino group but can be readily modifiedso as to have an amino group or an amino group-containing function isalso usable as the chelating compound (2) after the modification. Forinstance, one compound bearing a carboxyl group may be reacted withhexanediamine so that it is converted into one bearing anaminohexylaminocarbonyl group which can be readily condensed with aformyl group. Specific examples are diethylenetriamine-pentaacetic acid,ethylenediaminetriacetic acid,2-oxopropionaldehyde-bis(thiosemicarbazone) derivatives of the formula:##STR5## wherein R⁵, R⁶, R⁷ and R⁸ are each a hydrogen atom or a C₁ -C₃alkyl group [U.S. Pat. No. 4,287,362].

The term "physiologically active substance" as the constituent (3) isintended to mean any substance which shows a specific accumulability ata certain organ or tissue or a certain diseased locus or exhibits aspecific behavior corresponding to a certain physiological state.Tracing of the behavior of such substance in a living body can provideinformations useful for diagnosis. Physiologically active substanceshaving an amino group capable of being condensed with a formyl groupunder a relatively mild condition are advantageously useful in thisinvention. Even when an amino group is not present, however, thesubstance may be used as the physiologically active substance (3) aftera chemical modification so as to have an amino group or an aminogroup-containing function. Specific examples of suitable physiologicallyactive substances are blood proteins (e.g. human serum albumin,fibrinogen), enzymes (e.g. urokinase, streptokinase), hormones (e.g.thyroid stimulating hormone, parathyroid hormone), immune antibodies(e.g. IgG) , monoclonal antibodies, antibiotics (e.g. bleomycin,kanamycin), saccharides, fatty acids, amino acids, etc. In general, thisinvention is favorably applicable to physiologically active substanceshaving a molecular weight of not less than about 100,000.

The term "radioactive metallic element" as the constituent (4) isintended to mean any metallic element having radioactivity, a hasphysical characteristics suitable for nuclear medical diagnosis and canbe readily captured with the chelate forming structure in the chelatingcompound (2). Specific examples of the radioactive metallic element aregallium-67 (⁶⁷ Ga), gallium-68 (⁶⁸ Ga), thallium-201 (²⁰¹ T1),indium-111 (¹¹¹ In), tecnethium-99m (^(99m) Tc), etc. They are normallyemployed in their salt form, particularly in their water-soluble saltforms.

For preparation of the non-radioactive carrier of the invention, thepolyformyl compound (1) and the chelating compound (2) are subjected tocondensation to form a methyleneimine linkage between the formyl groupin the former and the the amino group in the latter, optionally followedby reduction of the methyleneimine linkage to the methyleneaminelinkage. Depending on the kinds of the reactants, the reactionconditions, etc., the number of the units of the chelating compound (2)to be introduced into the polyformyl compound (1) is varied, andgenerally a larger number of not less than about 5 units, especially ofnot less than about 10 units, of the chelating compound (2) per eachmolecule of the polyformyl compound (t) is better, but at least oneformyl group in the polyformyl compound (1) should be left forcombination with the physiologically active substance (3).

The resulting polyformyl compound (1) combined with the chelatingcompound (2) condensation or condensation-reduction product (hereinafterreferred to as "the condensation or condensation-reduction product") asthe non-radioactive carrier is then condensed with the physiologicallyactive substance (3), optionally followed by reduction so as to form amethyleneimine group or a methyleneamine group between a formyl group inthe polyformyl compound (1) moiety of the former and the amino group inthe latter to give the physiologically active substance-combinedcondensation or condensation-reduction product. The number of units ofthe physiologically active substance (3) to be introduced into thecondensation or condensation-reduction product varies with the kinds ofthe reactants and the reaction conditions, etc. The desirable number ofunits is usually less than about 10 units and is preferably not morethan 3 units of the physiologically active substance (3) per eachmolecule of the polyformyl compound (1).

Alternatively, the physiologically active substance-combinedcondensation or condensation-reduction product may be prepared by firstcondensing the polyformyl compound (1) with the physiologically activesubstance (3) to form a methyleneimine linkage between a formyl group inthe former and an amino group in the latter, optionally followed byreduction of the methyleneimine linkage to a methyleneamine linkage, togive the physiologically active substance-combined polyformyl compound.The compound is then condensed with the chelating compound (2) to form amethyleneimine linkage between a formyl group in the polyformyl compoundmoiety of the physiologically active substance-combined polyformylcompound and an amino group in the chelating compound (2), optionallyfollowed by reduction of the methyleneimine linkage to a methyleneaminelinkage, whereby the physiologically active substance-combinedcondensation or condensation-reduction product is obtained. As to thenumbers of the units of the chelating compound (2) and of thephysiologically active substance (3), the same attention as stated abovemay be taken.

In the above mentioned preparation procedures, the optional reductionafter the condensation may be accomplished in a single step at the finalstage. Further, each of the reactions such as condensation and reductionmay be carried out by conventional procedures. Furthermore, in thereduction, a formyl group may be converted into a hydroxymethyl groupsimultaneously with the conversion of a methyleneimine linkage into amethyleneamine linkage. Usually, the condensation can easily proceed atroom temperature. For the reduction, a reductive metal hydride compoundsuch as sodium borohydride is favorably employed as the reducing agent.

At any stage in the above preparation procedures, the reaction productmay be optionally purified by conventional methods such as columnchromatography, gel permeation and dialysis.

The resulting physiologically active substance-combined condensation orcondensation-reduction product may then be labeled with the radioactivemetallic element (4) to give a radioactive metallic element-labeled,physiologically active substance-combined condensation orcondensation-reduction product which is a radioactive diagnostic agentof the invention.

Depending upon the kind or state of the radioactive metallic element(4), two different labeling procedures may be adopted. When theradioactive metallic element (4) is in a valency state which can form astable chelate compound, the physiologically active substance-combinedcondensation or condensation-reduction product may be contacted with theradioactive metallic element (4) in an aqueous medium to form theradioactive metallic element-labeled physiologically activesubstance-combined condensation or condensation-reduction product as aradioactive diagnostic agent. This labeling manner may be applied to ⁶⁷Ga, ¹¹¹ In, etc. When the radioactive metallic element (4) is in avalency state which has to be changed for the formation of a stablechelate compound, the physiologically active substance-combinedcondensation or condensation-reduction product may be contacted with theradioactive metallic element (4) in an aqueous medium in the presence ofa reducing agent or an oxidizing agent to form the radioactive metallicelement-labeled, physiologically active substance-combined condensationor condensation-reduction product. This labeling manner may be appliedto ^(99m) Tc, etc.

Examples of the reducing agent are stannous salts, i.e. salts ofdivalent tin ion (Sn⁺⁺ ). Specific examples are stannous halides (e.g.stannous chloride, stannous fluoride), stannous sulfate, stannousnitrate, stannous acetate, stannous citrate, etc. Sn⁺⁺ ion-bearingresins, e.g. ion-exchange resins charged with Sn⁺⁺ ion, are alsosuitable.

When, for example, the radioactive metallic element (4) is ^(99m) Tc,the physiologically active substance-combined condensation orcondensation-reduction product may be treated with ^(99m) Tc in the formof a pertechnetate in an aqueous medium in the presence of a reducingagent, e.g. a stannous salt. There is no particular requirementconcerning the order of the introduction of the above reagents into thereaction system. Usually, however, initial mixing of the stannous saltwith the pertechnetate in an aqueous medium should be avoided. Thestannous salt may be used in an amount that can sufficiently reduce thepertechnetate.

The radioactive diagnostic agent should have sufficient radioactivityand radioactivity concentration to assure reliable diagnosis. Forinstance, the radioactive metallic element ^(99m) Tc may be used in anamount of 0.1 to 50 mCi in about 0.5 to 5.0 ml at the time ofadministration. The amount of the physiologically activesubstance-combined condensation or condensation-reduction product shouldbe sufficient to form a stable chelate compound with the radioactivemetallic element (4).

The resulting radioactive metallic element-labeled, physiologicallyactive substance-combined condensation or condensation-reduction productis sufficiently stable as a radioactive diagnostic agent, and thereforeit may be stored as such and supplied on demand. When desired, theradioactive diagnostic agent may contain any suitable additive such as apH controlling agent (e.g. an acid, a base, a buffer), a stabilizer(e.g. ascorbic acid) or an isotonizing agent (e.g. sodium chloride).

The radioactive metallic element-labeled, physiologically activesubstance-combined condensation or condensation-reduction product ofthis invention is useful for nuclear medical diagnosis. For example, the^(99m) Tc or ⁶⁷ Ga-labeled one may be used for recording and functionalmeasurement of myocardium. Also, the ^(99m) Tc-labeled human serumalbumin-combined condensation or condensation-reduction product can beused for recording, dynamic study and quantitative measurement of theblood circulation system by intravenous administration to the humanbody. Further, the ^(99m) Tc-labeled fibrinogen or urokinase-combinedcondensation or condensation-reduction product may be used for detectionand recording of thrombosis as well as the localization of thrombosis,since they accumulate at the locus of thrombosis. Furthermore, the^(99m) Tc-labeled streptokinase-combined condensation orcondensation-reduction product is useful for determination of the locusof a myocardial infarction. Moreover, the ^(99m) Tc-labeled thyroidstimulating hormone-combined condensation or condensation-reductionproduct is useful for the detection and recording of a cancer at thethyroid gland.

The radioactive diagnostic agent of this invention may be administeredto a patient in an amount sufficient to produce the radioactivitynecessary for examination of a particular organ or tissue, by anyappropriate route, but usually through an intravenous route. Forinstance, the intravenous administration of a ^(99m) Tc-labeledradioactive diagnostic agent in an amount of about 1 to 3 ml by volumehaving a radioactivity of about 1 to 20 mCi to a patient is quitesuitable for diagnostic purpose.

The advantages of the physiologically active substance-combinedcondensation or condensation-reduction product of this invention (i.e.the physiologically active substance-combined non-radioactive carrier)may be summarized as follows: (a) it is stable over a long period oftime after manufacture; (b) since it can be produced under mildconditions, no unfavorable side reactions such as inactivation,denaturation or decomposition are caused in the physiologically activesubstance; (c) any physiologically active substance having an aminogroup can be used as the starting material; (d) even when an amino groupis not present, the introduction of such group into a physiologicallyactive substance makes it usable as the starting material; (e) aradioactive metallic element-labeled, physiologically activecondensation or condensation-reduction product can be formed by a verysimple procedure (e.g. by merely contacting the physiologically activesubstance-combined condensation or condensation-reduction product with aradioactive metallic element in an aqueous medium). The advantages ofthe radioactive metallic element-labeled, physiologically activesubstance-combined condensation or condensation-reduction product usedas a radioactive diagnostic agent may be also summarized as follows: (a)it is stable over a long period of time after manufacture; (b) thelabeling efficiency with the radioactive metallic element is extremelyhigh (nearly 100 %); (c) since the labeling operation is quite simple,no unfavorable side reactions such as inactivation, denaturation ordecomposition are caused in the physiologically active substance bondedto the condensation or condensation-reduction product; (d) among variousradioactive metallic elements, the most suitable one for the diagnosticpurpose may be chosen so that the diagnosis can be improved not only inquantity but also in quality with reduction of the exposure dose.

Practical and presently preferred embodiments of the invention areillustratively shown in the following Examples.

REFERENCE EXAMPLE 1 Preparation of Polyacrolein

Water (50 ml) was charged in a flask and heated under reflux whileintroducing nitrogen gas therein. After cooling below 20° C. potassiumperoxodisulfate (0.475 g) and acrolein (purity, more than 95%) (10 ml)were added thereto. After acrolein was dissolved, a solution of silvernitrate (0.296 g) in water (6 ml) was dropwise added thereto in about 1minute with vigorous agitation. The reaction was continued for 2.5hours, during which care was taken to avoid raising the temperatureabove 20° C. After the reaction was completed, the reaction mixture wasadded to water (50 ml), whereby the polyacrolein produced wasprecipitated. The precipitate was collected by filtration, washed withwater two times and dispersed in a solution of sodium thiosulfate (0.5g) in water (50 ml), followed by stirring for 1 hour. The dispersion wasfiltered to collect the solid material, which was washed with waterseveral times and dried under reduced pressure overnight to obtainpolyacrolein,

Polyacrolein (50 mg) as prepared above was dissolved indimethylsulfoxide (10 ml), sodium borohydride (3 mg) was added thereto,and stirring was continued at room temperature for 1 hour. Ethyl acetate(10 ml) was added to the resulting mixture to precipitate partiallyreduced polyacrolein. The precipitate was collected by filtration,dissolved in water and subjected to measurement of molecular weight byhigh speed liquid chromatography under the following conditions:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH,7.4 )

Flow rate: 1.0 ml/min

Since the partially reduced polyacrolein was eluted at a retentionvolume of 23.2 ml, the molecular weight of polyacrolein was determinedto be about 21,000.

REFERENCE EXAMPLE 2 Preparation of Dialdehydrodextran

To a solution of dextran (average molecular weight, 10,000) (3.24 g) in0.1M sodium acetate solution (pH, 4 2; 200 ml), 0M sodium periodatesolution (40 ml) was added, and the resultant mixture was stirred in adark place overnight. The reaction mixture was placed into a cellulosetube and dialyzed to water for 2 days, followed by lyophilization toobtain dialdehydodextran.

About 50 mg of the above prepared dialdehydodextran was preciselyweighed and then dissolved in a 0.01M phosphoric acid-0.15M sodiumchloride buffer (100 ml). The resulting solution (about 5 ml) wasprecisely measured, 1/100 N iodine solution (5 ml) was added thereto,and 0.15M sodium carbonate solution (1 ml) was further added thereto,followed by allowing the solution to stand at room temperature for 1.5hours. After addition of 0.2N sulfuric acid (2 ml), titration wascarried out with 1/100N sodium thiosulfate solution until a colorless,transparent solution was obtained. This titration value was taken as A.In the same manner as above, 0.01M phosphoric acid-0.15M sodium chloridebuffer (5 ml) was titrated with 1/100N sodium thiosulfate solution, andthe resulting titration value was taken as B. The content of aldehydegroups in 1 mg of the product was calculated according to the followingequation:

Aldehyde groups (μmole/mg)=(A-B)×10/2W wherein W is the amount ofdialdehydrodextran (mg) contained in 5 ml of the sample. As the result,the aldehyde group content in the dialdehydrodextran as prepared abovewas determined to be 5.1 μmole/mg.

Example 1

(A) Preparation of the polyacrolein-deferoxamine condensation-reductionproduct as a non-radioactive carrier:

Polyacrolein (molecular weight, 21,000) (500 mg) was dissolved indimethylsulfoxide (10 ml), and the resultant solution was admixed with asolution of deferoxamine (420 mg) in dimethylsulfoxide (10 ml). Thereaction was continued at room temperature for 3 hours. Sodiumborohydride (100 mg) was added to the reaction mixture and stirring wascontinued at room temperature for 1 hour. The resultant mixture wassubjected to dialysis to water overnight, followed by gel chromatographyunder the following conditions:

Carrier: Sephadex G-50

Solvent: Water

Column: diameter, 4.5 cm; height, 50 cm

Flow rate: 2.5 ml/min

The polyarolein-deferoxamine condensation-reduction product was elutedat a volume of 270-400 ml, while the unreacted deferoxamine was elutedat a volume of 550 to 600 ml. The eluate containing thepolyacrolein-deferoxamine condensation-reduction product was subjectedto lyophilization.

The polyacrolein-deferoxamine condensation-reduction product thusobtained was dissolved in water, ferric chloride was added thereto, andthe resultant solution was analyzed by high speed liquid chromatographyunder the following conditions to determine a retention volume of 21.2ml:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH,7.4)

Flow rate: 1.0 ml/min

Absorptive wavelength: 420 nm

No free deferoxamine was detected. (The retention volume of deferoxaminein the above system is 32.8 ml.)

A definite amount of the polyacrolein-deferoxaminecondensation-reduction product as obtained above was dissolved in waterand a sufficient amount of an aqueous ferric chloride solution was addedthereto to make a 1:1 complex between the deferoxamine moiety in saidcondensation-reduction product and Fe(III) in said ferric chloride. Thereaction mixture was allowed to stand for 1 hour and then subjected tomeasurement of absorbance at 420 nm, whereby the number of thedeferoxamine moieties in said condensation-reduction product wasconfirmed to be 18.3 per one molecule of polyacrolein. The averagemolecular weight of said condensation-reduction product was thuscalculated to be about 32,000.

Deferoxamine and Fe(III) can still form a 1:1 complex having a maximumabsorption at 420 nm, and the ε_(max) value of the complex at 420 nm is2.63×10³.

Example 2

(A) Preparation of thepolyacrolein-hexanediamine:3-oxobutyral-bis(N-methylthiosemicarbazone)-carboxylicacid condensate condensation-reduction product as non-radioactivecarrier:

A solution of 3-oxobutyral-bis(N-methylthiosemicarbazone)carboxylic acid(hereinafter referred to as "KTS") (132 mg) in dry dioxane (5 ml) wascooled to about 10° C. Tri-n-butylamine (0.12 ml) and isobutylchloroformate (64 μl ) were added thereto. The resultant mixture wasstirred at the same temperature as above for about 50 minutes to obtaina mixed acid anhydride solution. A solution ofN-tert-butyloxycarbonyl-1,6-hexanediamine (104 mg) in dry dioxane (2 ml)was added to this solution and the resultant mixture was stirred at 10°C. for about 15 hours to produceN-tert-butyloxycarbonyl-1,6-hexanediamine:KTS condensate. A few drops ofconc. hydrochloric acid were added thereto to make a pH of about 2,whereby the N-tert-butyloxycarbonyl group was eliminated to give asolution of hexanediamine-KTS condensate.

The above solution was added to a solution of polyacrolein (200 mg) indimethylsulfoxide (5 ml), sodium borohydride (17.2 mg) was addedthereto, and the resultant mixture was reacted at room temperature for 3hours. The reaction mixture was subjected to dialysis by a conventionalprocedure for 30 hours to eliminate the unreacted reagents andlyophilized to obtain the polyacrolein-hexanediamine:KTS condensatecondensation-reduction product useful as a non-radioactive carrier.

The polyacrolein-hexanediamine:KTS condensate condensation-reductionproduct as above obtained was dissolved in water to make a concentrationof 3 mg/ml, and the resulting solution was subjected to an absorbancemeasurement at 334 nm using water as the control, whereby the number ofthe KTS moieties in said condensation-reduction product was confirmed tobe 21.3 per one molecule of polyacrolein. The average molecular weightof said condensation-reduction product was thus calculated to be about29,600.

The hexanediamine:KTS condensate still had a maximum absorption at 334nm, and its ε_(max) value was 4.37×10⁴.

(B) Preparation of the fibrinogen-combinedpolyacrolein-hexanediamine:KTS condensate condensation-reduction product(a fibrinogen-combined non-radioactive carrier):

A solution of the non-radioactive carrier obtained in (A) (beforelyophilization) (5 ml) was added to a solution of human fibrinogen (250mg) in a 0.01M phosphate buffer-0.15M aqueous sodium chloride mixture(pH, 8.4) (50 ml), followed by stirring at room temperature for about 3hours. Sodium borohydride (12.9 mg) was added thereto. The resultantmixture was stirred for about 1 hour. The reaction mixture was dialyzedto 0.01M glucose-0.35M sodium citrate solution at 0° to 4° C. for 24hours and then passed through a column of Sepharose 4B (diameter, 4.4cm; height, 50 cm) using 0.01M glucose-0.35M sodium citrate solution asan eluting solvent. The eluate was lyophilized to give thepolyacrolein-hexanediamine:KTS condensate condensation-reduction productas cotton-like crystals.

The cotton-like crystals (100 mg) were dissolved in deoxygenated water(160 ml), and 1 mM stannous chloride solution (10 ml) and sodiumascorbate. (0.6 g) were added thereto to make a clear Solution. Thesolution was passed through a filter having a pore diameter of 0.45 μm,and the filtrate (1.5 ml) was filled in a vial flushed with nitrogen gasto obtain a fibrinogen-combined non-radioactive carrier. The aboveoperations were effected under a sterile condition.

The fibrinogen-combined non-radioactive carrier as above obtained was apale yellow, clear solution.

(C) Preparation of the ^(99m) Tc-labeled, fibrinogen-combinedpolyacrolein-hexanediamine:KTS condensate condensation-reduction productas a radioactive diagnostic agent:

To the fibrinogen-combined non-radioactive carrier (1.5 ml) as obtainedin (B), a physiological saline solution (1.5 ml) containing ^(99m) Tc(3.3 mCi) in the form of sodium pertechnetate was added to obtain the^(99m) Tc-labeled, figrinogen-combined polyacrolein-hexanediamine:KTScondensate condensation-reduction product useful as a radioactivediagnostic agent.

This solution was pale yellow and transparent.

(D) Properties of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toelectrophoresis (1.7 mA/cm; 15 minutes) using Veronal buffer (pH, 8.6)as a developing solvent and a cellulose acetate membrane as anelectrophoretic membrane, and scanning was carried out by the use of aradiochromato-scanner. The radioactivity was recognized as a single peakat the locus of 0.5 cm distant from the original line towards thenegative side. This locus was the same as that of the coloring band offibrinogen with Ponceau 3R.

From the above result, it may be said that the radioactive diagnosticagent has a labeling efficiency of nearly 100% and its electric chargeis substantially the same as that of fibrinogen.

To the radioactive diagnostic agent as obtained in (C), 0.1M sodiumdiethylbarbiturate hydrochloride buffer (pH, 7.3) containing 0.05%calcium chloride was added to make a fibrinogen concentration of 1mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto. The resultantmixture was allowed to stand in an ice bath for 30 minutes. The producedfibrinogen clots were completely separated from the liquor, andradioactivity was measured on the clots and also on the liquor. From theobtained results, it was determined that the clottability of theradioactive diagnostic agent is 93% based on the starting fibrinogen.

Example 3

(A) Preparation of the polyacrolein-deferoxamine condensation product asa non-radioctive carrier:

To a solution of polyacrolein (125 mg) in dimethylsulfoxide (2.5 ml), asolution of deferoxamine (105 mg) in dimethylsulfoxide (2.5 ml) wasadded, and the resultant mixture was agitated at room temperature for 3hours to produce a solution containing the polyacrolein-deferoxaminecondensation product which is useful as a non-radioactive carrier.

(B) Preparation of the fibrinogen-combined, polyacrolein-deferoxaminecondensation product (a fibrinogen-combined non-radioactive carrier):

The non-radioactive carrier (5 ml) as obtained above was added to asolution of human fibrinogen (200 mg) in 0.01M phosphate buffer-0.15Maqueous sodium chloride mixture (pH, 8.4) at 0° to 4° C., followed bystirring at the same temperature as above for about 3 hours. Thereaction mixture was dialyzed to 0.01M glucose-0.35M sodium citratesolution at 0° to 4° C. for 24 hours and then passed through a column ofSepharose 4B (diameter, 4.4 cm; height, 50 cm) using 0.01M glucose-0.35Msodium citrate solution as an eluting solvent. The eluate containing thefibrinogen-combined polyacrolein-deferoxamine condensation product wasdiluted with 0.01M glucose-0.35M sodium citrate solution to make afibrinogen concentration of 1 mg/ml, and sodium ascorbate was addedthereto to make a concentration of 30 mM. The resultant solution (3 ml)was admitted into a vial, followed by lyophilization to obtain afibrinogen-combined non-radioactive carrier as a cotton-like product.The above operations were effected under a sterile condition.

Example 4

(A) Preparation of the polyacrolein-deferoxamine condensation product (anon-radioctive carrier):

To a solution of polyacrolein (125 mg) in dimethylsulfoxide (2.5 ml), asolution of deferoxamine (105 rag) in dimethylsulfoxide (2.5 ml) wasadded, and the resultant mixture was agitated at room temperature for 3hours to obtain a solution containing the polyacrolein-deferoxaminecondensation product useful as a non-radioactive carrier.

(B) Preparation of the fibrinogen-combined polyacrolein-deferoxaminecondensation-reduction product (a fibrinogen-combined non-radioactivecarrier):

The non-radioactive carrier (5 ml) as obtained above was added to asolution of human fibrinogen (200 mg) in 0.01M phosphate buffer-0.15Maqueous sodium chloride mixture (pH, 8.4) at 0° to 4° C., followed bystirring at the same temperature as above for about 3 hours. To theresulting mixture, sodium borohydride (7.0 mg) was added, and stirringwas continued at 0° to 4° C. for about 1 hour.

To a portion of the reaction mixture, a solution containing ⁶⁷ Ga (1mCi) in the form of gallium chloride was added for labeling, and theresultant solution was subjected to high speed liquid chromatographyunder the following conditions:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH7.4)

Pressure: 100 kg/cm²

Flow rate: 1.0 ml/min

Detection was made on the radioactivity of 67Ga. The resulting elutedpattern gave three peaks attributable to ⁶⁷ Ga-labeled fibrinogen, ⁶⁷Ga-labeled polyacrolein-deferoxamine condensation-reduction product and⁶⁷ Ga-labeled deferoxamine. From the area ratio of the peak due to ⁶⁷Ga-labeled polyacrolein-deferoxamine condensation-reduction product andthe peak due to Ga-labeled deferoxamine, 18.9 of the deferoxaminemoieties were confirmed to combine to one molecule of polyacrolein.Since the number of the deferoxamine moieties in the fibrinogen-combinedpolyacrolein-deferoxamine condensation-reduction product was confirmedto be 14.8 per one molecule of fibrinogen, the number of fibrinogenbonded to one molecule of polyacrolein was calculated to be about 0.8.

The remainder of the reaction mixture was dialyzed to 0.01Mglucose-0.35M sodium cintrate solution at 0° to 4° C. for 24 hours andthen passed through a column of Sepharose 4B (diameter, 4.4 cm; height,50 cm) as an eluting solvent. The eluate containing thefibrinogen-combined polyacrolein-deferoxamine condensation-reductionproduct was diluted with 0.01M glucose-0.35M sodium citrate solution tomake a fibrinogen concentration of 1 mg/ml, and sodium ascorbate wasadded thereto to make a concentration of 30 mM. The resultant solution(3 ml) was admitted into a vial, followed by lyophilization to obtain afibrinogen-combined non-radioactive carrier as a cotton-like product.The above operations were effected under a sterile condition.

The fibrinogen-combined non-radioactive carrier as obtained above wasdissolved in sterile water: to make a fibrinogen concentration of 1mg/ml, and a sufficient amount of an aqueous ferric chloride solutionwas added thereto to make a 1:1 complex between the deferoxamine moietyin said non-radioactive carrier and Fe(III) in said ferric chloridesolution. The reaction mixture was allowed to stand for 1 hour and thensubjected to measurement of absorbance at 420 nm using a solution ofsaid non-radioactive carrier in sterile water as the control, wherebythe number of the deferoxamine moieties in said non-radioactive carrierwas confirmed to be 14.8 per one molecule of fibrinogen.

(C) Preparation of the ⁶⁷ Ga-labeled, fibrinogen-combinedpolyacrolein-deferoxamine condensation-reduction product as aradioactive diagnostic agent:

To the fibrinogen-combined non-radioactive carrier as obtained in (B),an aqueous solution (2 ml) containing ⁶⁷ Ga (2 mCi) in the form ofgallium citrate was added to obtain the ⁶⁷ Ga-labeled,fibrinogen-combined polyacrolein-deferoxamine condensation-reductionproduct as a radioactive diagnostic agent.

This solution was pale yellow, transparent and had a pH of about 7.8.

(C') Preparation of the ⁶⁷ Ga-labeled, fibrinogen-combinedpolyacrolein-deferoxamine condensation-reduction product as aradioactive diagnostic agent:

The fibrinogen-labeled non-radioactive carrier obtained in (B) wasdissolved in sterile water, and human fibrinogen (0.5, 0.75, 1.0, 1.5,2.0 or 3.0 mg) dissolved in 0.01M phosphate buffer-0.15M aqueous sodiumchloride mixture (pH, 8.4) and 1 ml of an aqueous solution containing ⁶⁷Ga (1 mCi) in the form of gallium citrate were added thereto. Theresulting mixture was allowed to stand at room temperature for 1 hourand then subjected to measurement of lebeling rate. In the same manneras above, the labeling rate of ⁶⁷ Ga-labeled, fibrinogen-combineddeferoxamine as prepared by labeling ⁶⁷ Ga onto fibrinogen-combineddeferoxamine was also measured. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        (Labeling efficiency with .sup.67 Ga)                                                      Labeling rate (%)                                                Fibrinogen (mg)                                                                              Sample 1*.sup.1)                                                                         Sample 2*.sup.2)                                    ______________________________________                                        0.5            59.3       --                                                   0.75          83.2       --                                                  1.0            97.8       17.0                                                1.5            ˜100 --                                                  2.0            ˜100 35.2                                                3.0            ˜100 --                                                  6.3            --         41.4                                                12.6           --         70.9                                                18.8           --         80.6                                                25.1           --         83.5                                                ______________________________________                                         Note:                                                                         *.sup.1) Radioactive diagnostic agent according to the invention.             *.sup.2) .sup.67 Galabeled fibrinogencombined deferoxamine               

As understood from the above, the non-radioactive carrier of theinvention could be labeled with 97.8% of ⁶⁷ Ga (1 mCi) within 1 hourwhen 1 mg of fibrinogen was used. The conventional non-radioactivecarrier (i.e. fibrinogen-combined deferoxamine) could be labeled onlywith 17.0% of ⁶⁷ Ga under the same condition as above. Even when 25.1 mgof fibrinogen were used, the conventional non-radioactive carrier waslabeled with 83.5% of ⁶⁷ Ga at the most. It is thus appreciated that thenon-radioactive carrier of the invention can afford a radioactivediagnostic agent having a higher relative radioactivity. Further, theradioactive diagnostic agent is useful in nuclear medical diagnosisaiming at detection of thrombosis.

(D) Properties of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toelectrophoresis (1.7 mA/cm; 15 minutes) using Veronal buffer (pH, 8.6)as a developing solvent and a Cellulose acetate membrane as anelectrophoretic membrane, and scanning was carried out by the use of aradiochromato-scanner. The radioactivity was recognized as a single peakat the locus of 0.5 cm distant from the original line towards thenegative side. This locus was the same as that of the coloring band offibrinogen with Ponceau 3R.

From the above result, it may be said that the radioactive diagnosticagent has a labeling efficiency of nearly 100% and its electric chargeis substantially the same as that of fibrinogen.

To the radioactive diagnostic agent as obtained in (C), 0.1M sodiumdiethylbarbiturate hydrochloride buffer (pH, 7.3 ) containing 0.05%calcium chloride was added to make a fibrinogen concentration of 1mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto. The resultantmixture was allowed to stand in an ice bath for 30 minutes. The producedfibrinogen clots were completely separated from the liquor, andradioactivity was measured on the clots and also on the liquor. From theresults obtained, it was determined that the clottability of theradioactive diagnostic agent is 86% based on the starting fibrinogen.

(E) Behaviors of the radioactive diagnostic agent as obtained in (C) inrats:

The radioactive diagnostic agent as obtained in (C) (0.2 ml) wasadministered intravenously to each of the female rats of SD strain, andthe variations of the blood level and the organ distribution with thelapse of time were recorded. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        (Distribution in rat body; %/g)                                                           Time after administration (min)                                   Organs        5      30        60   180                                       ______________________________________                                        Blood         8.33   6.82      6.22 4.81                                      Liver         1.47   1.62      1.73 1.78                                      Heart         0.85   0.88      1.03 0.96                                      Spleen        1.21   1.15      1.32 1.34                                      Large intestine                                                                             0.11   0.18      0.15 0.20                                      Small intestine                                                                             0.24   0.35      0.36 0.41                                      ______________________________________                                    

The extremely high blood level over a long period of time and the figureof distribution into various organs of the radioactive diagnostic agentas shown in Table 2 are quite similar to those of ¹³¹ I-labeledfibrinogen as conventionally employed.

(F) Behaviors of the radioactive diagnostic agent as obtained in (C) inthrombosed rabbits:

Thrombosis was produced in rabbits at the femoral part by the formalinapplication procedure. The radioactive diagnostic agent (0.5 ml) asobtained in (C) was administered to the rabbits through an ear vein.After 2.4 hours from the administration, a constant amount of the bloodwas sampled and the locus of thrombosis was taken out. Radioactivity wasmeasured on the blood and the locus of thrombosis. The radioacitivityratio of the locus of thrombosis to the blood for the same weight was7.44±3.41 (average in 10 animals±S.D. value).

From the above results, it is understood that the radioactive diagnosticagent as obtained in (C) has the nearly same physiological activity asdoes fibrinogen. Thus, the radioactive diagnostic agent is useful fornuclear medical diagnosis.

(G) Toxicity of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toattenuation of the radioactivity to an appropriate extent, and theresultant product was administered intravenously to groups of male andfemale rats of SD strain. Each group consisted of five animals, and adose of 1 ml per 100 grams of the bodyweight (corresponding to 600 timesthe expected dose to human beings) was administered. Also, groups ofmale and female mice of ICR strain, each group consisting of fiveanimals, a dose of 0.5 ml per 1.0 gram of the bodyweight (correspondingto 3,000 times the expected dose to human beings) was administered. Asthe control, the same volume of a physiological saline solution as abovewas intravenously administered to separate groups of the same animals asabove. The animals were fertilized for 10 days, and the variation inbodyweight during that period was recorded. No significant differencewas recognized between the medicated groups and the control groups.

After 10 days from the administration, all the animals were sacrificedand subjected to observation of the abnormality in various organs. But,no abnormality was seen in any of the animals.

From the above results, it may be said that the toxicity of thenon-radioactive carrier of the invention is extremely low.

Example 5

(A) Preparation of the dialdehydostarch-deferoxamine condensationproduct as a non-radioactive carrier:

Dialdehydostarch (average molecular weight, 7000; oxidation rate, 80%)(1 g) was dissolved in water (40 ml). Separately, deferoxamine (2.4 g)was dissolved in water (30 ml), an equimolar amount of triethylamine(388 mg) was added thereto, and the resultant solution was stirred atroom temperature for 10 minutes. Both solutions were combined togetherand stirred at room temperature for 15 minutes. The reaction mixture wassubjected to gel chromatography under the following conditions:

Carrier: Sephadex G-50

Solvent: Water

Column: diameter, 4.5 cm; height, 50 cm

Flow rate: 2.5 ml/min

The dialdehydostarch-deferoxamine condensation product was eluted at avolume of 270-430 ml, while the unreacted deferoxamine was eluted at avolume of 550 to 600 ml. The eldate containing thedialdehydostarch-deferoxamine condensation product was subjected tolyophilization.

The dialdehydostarch-deferoxamine condensation product thus obtained wassubjected to analysis by high speed liquid chromatography under thefollowing conditions:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH,7.4)

Pressure: 100 kg/cm²

Flow rate: 1.0 ml/min

Absorptive wavelength: 280 nm

No free deferoxamine was detected. (The retention volume of deferoxaminein the above system is 32.8 ml.)

Example 6

(A) Preparation of the dialdehydostarch-deferoxaminecondensation-reduction product as a non-radioactive carrier:

Dialdehydostarch (average molecular weight, 7000; oxidation rate, 80%)(1 g) was dissolved in water (40 ml). Separately, deferoxamine (2.4 g)was dissolved in water (30 ml), an equimolar amount of triethylamine(388 mg) was added thereto, and the resultant solution was stirred atroom temperature for 10 minutes. Both solutions were combined togetherand stirred at room temperature for 15 minutes. Sodium borohydride (167mg) was added thereto, and stirring was continued at room temperaturefor about 1 hour. The reaction mixture was subjected to gelchromatography under the following conditions:

Carrier: Sephadex G-50

Solvent: Water

Column: diameter, 4.5 cm; height, 50 cm

Flow rate: 2.5 ml/min

The dialdehydostarch-deferoxamine condensation-reduction product waseluted at a volume of 300-450 ml, while the unreacted deferoxamine waseluted at a volume of 550 to 600 ml. The eluate containing thedialdehydostarch-deferoxamine condensation-reduction product wassubjected to lyophilization.

The dialdehydostarch-deferoxamine condensation reduction product thusobtained was subjected to analysis by high speed liquid chromatographyunder the following conditions:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH,7.4 )

Pressure: 100 kg/cm²

Flow rate: 1.0 ml/min

Absorptive wavelength: 280 nm

No free deferoxamine was detected. (The retention volume of deferoxaminein the above system is 32.8 ml.)

A definite amount the dialdehydostarch-deferoxaminecondensation-reduction product was dissolved in water, and a sufficientamount of an aqueous ferric chloride solution was added thereto to makea 1:1 complex between the deferoxamine moiety in saidcondensation-reduction product and Fe(III) in said ferric chloridesolution. The reaction mixture was allowed to stand for 1 hour and thensubjected to measurement of absorbance at 420 nm, whereby it wasconfirmed that the number of the deferoxamine moieties in saidcondensation-reduction product is. 19.6 per one molecule ofdialdehydostarch. The number average molecular weight of saidcondensation-reduction product was thus calculated to be about 18,000.

Still, deferoxamine and Fe(III) can form a 1:1 complex having a maximumabsorption at 420 run, and the ε_(max) value of the complex at 420 nm is2.63×10³.

Example 7

(A) Preparation of the dialdehydostarch-hexanediamine:KTS condendatecondensation-reduction product as a non-radioactive carrier:

A solution of KTS (132 mg) in dry dioxane (5 ml) was cooled to about 10°C. Tri-n-butylamine (0.12 ml) and isobutyl chloroformate (64 ml) wereadded thereto. The resultant mixture was stirred at the same temperatureas above for about 50 minutes to obtain a mixed acid anhydride solution.To this solution, a solution ofN-tert-butyloxy-carbonyl-1,6-hexanediamine (104 mg) in dry dioxane (2ml) was added, and the resultant mixture was stirred at 10° C. for about15 hours to produce N-tert-butyioxycarbonyl-1,6-hexanediamine:KTScondensate. A few drops of conc. hydrochloric acid were added thereto tomake a pH of about 2, whereby the N-tert-butyloxycarbonyl group waseliminated to give a solution of hexanediamine-KTS condensate.

The above solution was added to a solution of dialdehydostarch (200 mg)in dimethylsulfoxide (5 ml), sodium borohydride (17.2 mg) was addedthereto, and the resultant mixture was reacted at room temperature for 3hours. The reaction mixture was subjected to dialysis by a conventionalprocedure for 30 hours to eliminate the unreacted reagents andlyophilized to obtain dialdehydostarch-hexanediamine:KTS condensatecondensation-reduction product.

The dialdehydrostarch-hexanediamine:KTS condensatecondensation-reduction product as obtained above was dissolved in waterto make a concentration of 3 mg/ml, and the resulting solution wassubjected to measurement of absorbance at 334 nm using water as thecontrol, whereby it was confirmed that the number of the KTS moieties insaid condensation-reduction product is 11.2 per one molecule ofdialdehydostarch. The average molecular-weight of saidcondensation-reduction product was thus calculated to be about 11,500.

The hexanediamine:KTS condensate still had a maximum absorption at 334nm, and its ε_(max) value was 4.37×10⁴.

(B) Preparation of the fibrinogen-combineddialdehydostarch-hexanediamine:KTS condensate condensation-reductionproduct (a fibrinogen-combined non-radioactive carrier):

The hexanediamine:KTS condensate solution as obtained in (A) was addedto a solution of dialdehydostarch (200 mg) in dimethylsulfoxide (5 ml),and the resultant mixture was stirred at room temperature for about 3hours. The resulting solution containing thedialdehydrostarch-hexanediamine:KTS condensate condensation product (5ml ) was added to a solution of fibrinogen (250 mg) in 90.01M phosphatebuffer-0.15M aqueous sodium chloride mixture (pH, 8.4) (50 ml), followedby stirring at room temperature for about 3 hours. Sodium borohydride(12.9 mg) was added thereto. The resultant mixture was stirred for about1 hour. The reaction mixture was dialyzed to 0.01M glucose-0.35M sodiumcitrate solution at 0° to 4° C. for 24 hours and then passed through acolumn of Sepharose 4B (diameter, 4.4 cm; height, 50 cm) using 0.01Mglucose-0.35M sodium citrate solution as an eluting solvent. The eluatewas lyophilized to give the dialdehydostarch-hexanediamine:KTScondensate condensation reduction product as cotton-like crystals. Thecotton-like crystals (100 mg) were dissolved in deoxygenated water (160ml), and 1 mM stannous chloride Solution (10 ml) and sodium ascorbate(0.6 g) were added thereto to make a clear solution.. The solution waspassed through a filter having a pore diameter of 0.22 μm, and thefiltrate (1.5 ml) was filled in a vial flushed with nitrogen gas toobtain a fibrinogen-combined non-radioactive carrier as a pale yellow,transparent solution. The above operations were effected under a sterilecondition.

(C) Preparation of the ^(99m) Tc-labeled, fibrinogen-combineddialdehydostarch-hexanediamine:KTS condensate condensation-reductionproduct as a radioactive diagnostic agent:

To the fibrinogen-combined non-radioactive carrier (1.5 ml) as obtainedin (B), there was added a physiological saline solution (1.5 ml)containing ^(99m) Tc (3.3 mCi) in the form of sodium pertechnetate,followed by stirring for 15 minutes to obtain the ^(99m) Tc-labeled,figrinogen-combined polyacrolein-hexanediamine:KTS condensatecondensation-reduction product useful as a radioactive diagnostic agent.This solution was pale yellow and transparent.

(D) Properties of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toelectrophoresis (1.7 mA/cm; 15 minutes) using a Veronal buffer (pH, 8.6)as a developing solvent and a cellulose acetate membrane as anelectrophoretic membrane, and scanning was carried out by the use of aradiochromato-scanner. The radioactivity was recognized as a single peakat the locus of 0.5 cm distant from the original line towards thenegative side. This locus was the same as that of the coloring band offibrinogen with Ponceau 3R.

From the above result, it may be said that the radioactive diagnosticagent as obtained in (C) has a labeling efficiency of nearly 100% andits electric charge is substantially the same as that of fibrinogen.

To the radioactive diagnostic agent as obtained in (C), 0.1 M Sodiumdiethylbarbiturate hydrochloride buffer (pH, 7.3) containing 0.05%calcium chloride to make a fibrinogen concentration of 1 mg/ml. Thrombin(100 units/ml; 0.1 ml) was added thereto. The resultant mixture wasallowed to stand in an ice bath for 30 minutes. The produced fibrinogenclots were completely separated from the liquor, and radioactivity wasmeasured on the clots and also on the liquor. From the obtained results,it was determined that the clottability of the radioactive diagnosticagent is 91% based on the starting fibrinogen.

Example 8

(A) Preparation of the dialdehydrostarch-deferoxamine condensationproduct as a non-radioactive carrier:

To a solution of deferoxamine (130 mg) in 0.01M phosphoric acid-0.15Msodium chloride buffer (1.5 ml), triethylamine (99% solution; 27.9 ul)was added, and the resultant mixture was agitated at room temperaturefor 5 minutes. An aqueous solution of dialdehydrostarch (25 mg/ml; 2 ml)was added thereto. The resulting mixture was stirred at room temperaturefor 15 minutes to obtain a solution containing thedialdehydrostarch-deferoxamine condensation product which is useful as anon-radioactive carrier.

(B) Preparation of the fibrinogen-combined dialdehydostarch-deferoxaminecondensation product fibrinogen-combined non-radioactive carrier):

The non-radioactive carrier (5 ml) as obtained in (A) was added to asolution of human fibrinogen (200 mg) in 0.01M phosphate buffer-0.15Maqueous sodium chloride mixture (pH, 8 4) (30 ml) at 0° to 4° C.followed by stirring at the same temperature as above for about 3 hours.The reaction mixture was dialyzed to 0.01M glucose-0.35M sodium citratesolution at 0° to 4° C. for 24 hours and then passed through a column ofSepharose 4B (diameter, 4.4 cm; height, 50 cm) using 0.01M glucose-0.35Msodium citrate solution as an eluting solvent.

The eluate containing the fibrinogen-combineddialdehydostarch-deferoxamine condensation product was diluted with0.01M glucose-0.35M sodium citrate solution to make a fibrinogenconcentration of 1 mg/ml, and sodium ascorbate was added thereto to makea concentration of 30 mM. The resultant solution (3 ml) was admittedinto each vial, followed by lyophilization to obtain afibrinogen-combined, non-radioactive carrier as a cotton-like product.The above operations were effected under a sterile condition.

Example 9

(A) Preparation of the dialdehydostarch-deferoxamine condensationproduct as a non-radioctive carrier:

To a solution of deferoxamine (130 mg) in 0.01M phosphate buffer-0.15Maqueous sodium chloride solution (1.5 ml), triethylamine (99% solution)(27.9 μl) was added, and the resultant mixture was agitated at roomtemperature for 5 minutes. An aqueous solution of dialdehydrostarch (25mg/ml; 2 ml) was added thereto, and stirring was continued at roomtemperature for 15 minutes to obtain a solution containing thedialdehydostarch-deferoxamine condensation product which is useful as anon-radioactive carrier.

(B) Preparation of the fibrinogen-combined dialdehydostarch-deferoxaminecondensation-reduction product (a fibrinogen-combined non-radioactivecarrier):

The non-radioactive carrier (3.5 ml) as obtained above was added to asolution of fibrinogen (200 mg) in 0.01M phosphate-0.15M aqueous sodiumchloride mixture (pH, 8.4) (30 ml) at 0° to 4° C., followed by stirringat the same temperature as above for about 3 hours. To the resultingmixture, sodium borohydride (12.9 mg) was added, and stirring wascontinued at 0° to 4° C. for about 1 hour for reduction.

To a portion of the reaction mixture, a solution of gallium citratecontaining ⁶⁷ Ga (1 mCi) was added for labeling, and the resultantsolution was subjected to high speed liquid chromatography under thefollowing conditions:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH7.4)

Pressure: 100 kg/cm²

Flow rate: 1.0 ml/min.

Detection was made on the radioactivity of ⁶⁷ Ga. As the result, aeluted pattern gave three peaks attributable to ⁶⁷ Ga-labeledfibrinogen, the ⁶⁷ Ga-labeled dialdehydrostarch-deferoxaminecondensation-reduction product and ⁶⁷ Ga-labeled-deferoxamine. From thearea ratio of the peak due to ⁶⁷ Ga-labeleddialdehydrostarch-deferoxamine condensation reduction product and thepeak due to ⁶⁷ Ga-labelled deferoxamine, it was confirmed that 17.4 ofthe deferoxamine moieties are combined to one molecule ofdialdehydostarch. Since the number of deferoxamine moieties in thefibrinogen-combined dialdehydrostarch-deferoxaminecondensation-reduction product was confirmed to be 15.2 per one moleculeof fibrinogen, the number of fibrinogen bonded to one molecule ofdialdehydrostarch was calculated as about 0.9.

The remainder of the reaction mixture was dialyzed to 0.01Mglucose-0.35M sodium cintrate solution at 0° to 4° C. for 24 hours andthen passed through a column of Sepharose 4B (diameter, 4.4 cm; height,50 cm) using the same solution as above as an eluting solvent. Theeluate containing the fibrinogen-combined dialdehydostarch-deferoxaminecondensation-reduction product was diluted with 0.01M glucose-0.35Msodium citrate solution to make a fibrinogen concentration of 1 mg/ml,and sodium ascorbate was added thereto to make a concentration of 30 mM.The resultant solution (3 ml) was filled in a vial, followed bylyophilization to obtain a fibrinogen-combined non-radioactive carrieras a cotton-like product. The above operations were effected under asterile condition.

The fibrinogen-combined non-radioactive carrier was dissolved in sterilewater to make a fibrinogen concentration of 1 mg/ml, and a sufficientamount of an aqueous ferric chloride solution was added thereto to makea 1:1 complex between the deferoxamine moiety in said non-radioactivecarrier and Fe(III) in said ferric chloride solution. The reactionmixture was allowed to stand for 1 hour and then subjected to anabsorbance measurement at 420 nm using a solution of saidnon-radioactive carrier in sterile water as control, whereby the numberof the deferoxamine moieties in said non-radioactive carrier wasconfirmed to be 15.2 per one molecule of fibrinogen.

(C) Preparation of the ⁶⁷ Ga-labeled, fibrinogen-combineddialdehydostarch-deferoxamine condensation-reduction product as aradioactive diagnostic agent:

To the fibrinogen-combined non-radioactive carrier as obtained in (B),an aqueous solution (2 ml) containing ⁶⁷ Ga (2 mCi) in the form ofgallium citrate was added to obtain the ⁶⁷ Ga-labeled,fibrinogen-combined dialdehydo-starch deferoxaminecondensation-reduction product as a radioactive diagnostic agent. Thissolution was pale yellow, transparent and had a pH of about 7.8.

(C')- Preparation of the ⁶⁷ Ga-labeled, fibrinogen-combineddialdehydostarch-deferoxamine condensation-reduction product as aradioactive diagnostic agent:

The non-radioactive carrier obtained in (B) was dissolved in sterilewater, and human fibrinogen (0.5, 0.75, 1.0, 1.5, 2.0 or 3.0 mg)dissolved in 0.01M phosphoric acid-0.15M sodium chloride buffer (pH,8.4) and 1 ml of an aqueous solution containing ⁶⁷ Ga (1 mCi) in theform of gallium citrate were added thereto. The resulting mixture wasallowed to stand at room temperature for 1 hour and then subjected tomeasurement of the labeling rate. In the same manner as above, thelabeling rate of ⁶⁷ Ga-labeled, fibrinogen-combined deferoxamine asprepared by labeling ⁶⁷ Ga onto fibrinogen-combined deferoxamine wasalso measured. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        (Labeling efficiency with .sup.67 Ga)                                                      Labeling rate (%)                                                Fibrinogen (mg)                                                                              Sample 1*.sup.1)                                                                         Sample 2*.sup.2)                                    ______________________________________                                        0.5            68.4       --                                                   0.75          85.4       --                                                  1.0            ˜100 17.0                                                1.5            ˜100 --                                                  2.0            ˜100 35.2                                                3.0            --         --                                                  6.3            --         41.4                                                12.6           --         70.9                                                18.8           --         80.6                                                25.1           --         83.5                                                ______________________________________                                         Note:                                                                         *.sup.1) Radioactive diagnostic agent according to the invention.             *.sup.2) .sup.67 Galabeled fibrinogencombined deferoxamine               

As understood from the above, the non-radioactive carrier of theinvention could be labeled with 100% of ⁶⁷ Ga (1 mCi) within 1 hour when1 mg of fibrinogen was used. The conventional non-radioactive carrier(i.e. fibrinogen-combined deferoxamine) could be labeled only with 17.0%of ⁶⁷ Ga under the same condition as above. Even when 25.1 mg offibrinogen were used, the conventional non-radioactive carrier islabeled with 83.5% of ⁶⁷ Ga at most. It is thus appreciated that thenon-radioactive carrier of the -invention can afford a radioactivediagnostic agent having a higher relative radioactivity. Further, theradioactive diagnostic agent is useful in nuclear medical diagnosisaiming at detection of thrombosis.

(D) Properties of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toelectrophoresis (1.7 mA/cm; 15 minutes) using a Veronal buffer (pH, 8.6)as a developing solvent and a cellulose acetate membrane as anelectrophoretic membrane, and scanning was carried out by the use of aradiochromato-scanner. The radioactivity was recognized as a single peakat the locus of 0.5 cm distant from the original line towards thenegative side. This locus was the same as that of the coloring band offibrinogen with Ponceau 3R.

From the above result, it may be said that the radioactive diagnosticagent as obtained in (C) has a labeling efficiency of nearly 100% andits electric charge is substantially the same as that of fibrinogen.

To the radioactive diagnostic agent as obtained in (C), a 0.1M sodiumdiethylbarbiturate hydrochloride buffer (pH, 7.3) containing 0.05%calcium chloride was added to make a fibrinogen concentration of 1mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto. The resultantmixture was allowed to stand in an ice bath for 30 minutes. The producedfibrinogen clots were completely separated from the liquor, andradioactivity was measured on the clots and also on the liquor. From theobtained results, it was determined that the clottability of theradioactive diagnostic agent is 89% based on the starting fibrinogen.

(E) Behaviors of the radioactive diagnostic agent obtained in (C) inrats:

The radioactive diagnostic agent as obtained in (C) (0.2 ml) wasadministered intravenously to each of female rats of SD strain, and thevariations of the blood level and the organ distribution with the lapseof time were recorded. The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        (Distribution in rat body; %/g)                                                           Time after administration (min)                                   Organs        5      30        60   180                                       ______________________________________                                        Blood         8.74   7.08      6.62 5.34                                      Liver         1.45   1.32      1.05 1.03                                      Heart         0.90   0.89      1.40 0.98                                      Spleen        0.92   0.52      1.89 0.84                                      Large intestine                                                                             0.18   0.11      0.17 0.29                                      Small intestine                                                                             0.25   1.75      0.44 0.45                                      ______________________________________                                    

The extremely high blood level over a long period of time and the figureof distribution into various organs of the radioactive diagnostic agentas shown in Table 4 are quite similar to those of ¹³¹ I-labeledfibrinogen as conventionally employed.

(F) Behaviors of the radioactive diagnostic agent obtained in (C) inthrombosed rabbits:

Thrombosis was produced in rabbits at the femoral part by the formalinapplication procedure. The radioactive diagnostic agent (0.5 ml)obtained in (C) was administered to the rabbits through the ear vein.After 24 hours from the intravenous administration, a constant amount ofthe blood was sampled, and the locus of thrombosis was taken out.Radioactivity was measured on the blood and the locus of thrombosis. Theradioacitivity ratio of the locus of thrombosis to the blood for thesame weight was 8.63±3.83 (average in 10 animals±S.D. value).

From the above results, it is understood that the radioactive diagnosticagent obtained in (C) has the nearly same physiological activity asfibrinogen does. Thus, the radioactive diagnostic agent is useful fornuclear medical diagnosis.

(G) Toxicity of the radioactive diagnostic agent obtained in (C):

The radioactive diagnostic agent obtained in (C) was subjected toattenuation of the radioactivity to an appropriate extent, and theresultant product was administered intravenously to groups of male andfemale rats of SD strain. Each group consisted of five animals, and adose of 1 ml per 100 grams of the bodyweight (corresponding to 600 timesthe expected dose to human beings) was injected. Also groups of male andfemale mice of ICR strain, each group consisting of five animals, a doseof 0.5 ml per 1.0 gram of the bodyweight (corresponding to 3,000 timesthe expected dose to human beings) was administered. As the control, thesame volume of a physiolcgical saline solution as above wasintravenously administered to the separate groups of the same animals asabove. The animals were fertilized for 10 days, and the variation inbodyweight during that period was recorded. No significant differencewas recognized between the medicated groups and the control groups.

After 10 days from the administration, all the animals were sacrificedand subjected to observation of the abnormality in various organs. Noabnormality was seen in any of the animals.

From the above results, it may be said that the toxicity of thenon-radioactive carrier of the invention is extremely low.

EXAMPLE 10

(A) Preparation of dialdehydodextran-deferoxamine condensation-reductionproduct as a non-radioactive carrier:

To a solution of deferoxamine (2.8 g) in water (30 ml), an equimolaramount of triethylamine (432 mg) was added, followed by stirring at roomtemperature for 10 minutes. The resultant solution was added to asolution of dialdehydodextran (1 g; aldehyde group content, 5.1μmole/mg) in water (40 ml), followed by stirring at room temperature for15 minutes. To the reaction mixture, sodium borohydride (167 mg) wasadded, and stirring was continued at room temperature for about 1 hour.The resulting solution was admitted in a cellulose tube and dialyzed towater for 3 days, followed by gel chromatography under the followingconditions:

Carrier: Sephadex G-50

Solvent: water

Column: diameter, 4.5 cm; height, 50 cm

Flow rate: 2.5 ml/min

The dialdehydodextran-deferoxamine condensation-reduction product waseluted at a volume of 300 to 450 ml, while the unreacted deferoxaminewas eluted at a volume of 550 to 600 ml. The eluate containing saidcondensation-reduction product was lyophilized.

The lyophilized product was subjected to analysis with high speed liquidchromatography under the following conditions:

Column: TSK-3000SW

Solvent: 0.05M Tris-0.15M sodium chloride-hydrochloric acid buffer (pH,7.4)

Pressure: 100 kg/cm²

Flow rate: 1.0 ml/min

Absorptive wavelength: 280 nm

As a result, said condensation-reduction product was confirmed to show aretention volume of 27.3 ml. No free deferoxamine was detected. (Theretention volume of deferoxamine in the above system is 32.8 ml.)

(B) Preparation of the fibrinogen-combineddialdehydrodextran-deferoxamine condensation-reduction product (afibrinogen-combined, non-radioctive carrier):

Into a solution of dialdehydodextran (127 mg) a 90.01M phosphatebuffer-0.15M aqueous sodium chloride mixture (5 ml), deferoxamine (370mg) was dissolved, and triethyamine (78.9 μ) was added thereto, followedby stirring at 10° to 15° C. for 20 minutes. The resulting solution wasadded to a solution of fibrinogen (400 mg) in 90.01M phosphatebuffer-0.15M aqueous sodium chloride solution (40 ml) at 10° to 15° C.,followed by stirring at the same temperature as above for about 2 hours.To the reaction mixture, sodium borohydride (12.3 mg) was added, andstirring was continued at 10° to 15° C. for about 1 hour.

The resulting mixture was dialyzed to 0.01M glucose-0.35M sodium citratesolution at 0° to 4° C. for 3 days and then passed through a column ofSepharose CL6B (diameter, 4.4 cm; height, 100 cm) using the samesolution as above as a eluting solvent. The eluate was diluted with0.01M glucose-0.35M sodium citrate solution to make a fibrinogenconcentration of 1 mg/ml, and sodium ascorbate was added to make aconcentration of 30 mM. The resultant solution (3 ml) was filled in avial, followed by lyophilization to obtain a cotton-like product, whichis useful as a fibrinogen-combined non-radioactive carrier. The aboveoperations were effected under a sterile condition.

(C) Preparation of the ⁶⁷ Ga-labeled, fibrinogen-combineddialdehydodestran-deferoxamine condensation-reduction product as aradioactive diagnostic agent:

To the fibrinogen-combined non-radioactive carrier obtained in (B), anaqueous solution (2 ml) containing ⁶⁷ Ga (2 mCi) in the form of galliumcitrate was added to obtain the ⁶⁷ Ga-labeled, fibrinogen-combineddiaidehydostarch deferoxamine condensation-reduction product useful as areadioactive diagnostic agent. This solution was pale yellow,transparent and had a pH of about 7.8.

(D) Properties of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toelectrophoresis (1.7 mA/cm; 15 minutes) using a Veronal buffer (pH, 8.6)as a developing solvent and a cellulose acetate membrane as anelectrophoretic membrane, and scanning was carried out by the use of aradiochromato-scanner. The radioactivity was recognized as a single peakat the locus of 0.5 cm distant from the original line towards thenegative side. This locus was the same as that of the coloring band offibrinogen with Ponceau 3R.

From the above result, it may be said that the ⁶⁷ Ga-labeled,fibrinogen-combined dialdehydodextran-deferoxaminecondensation-reduction product has a labeling efficiency of nearly 100%and its electric charge is substantially the same as that of humanfibrinogen.

To the radioactive diagnostic agent as obtained in (C), 0.1M sodiumdiethylbarbiturate hydrochloride buffer (pH, 7.3) containing 0.05%calcium chloride was added to make a fibrinogen concentration of 1mg/ml. Thrombin (100 units/ml; 0.1 ml) was added thereto. The resultantmixture was allowed to stand in an ice bath for 30 minutes. The producedfibrinogen clots were completely separated from the liquor, andradioactivity was measured on the clots and also on the liquor. From theobtained results, it was determined that the clottability of theradioactive diagnostic agent is 84% based on the starting fibrinogen.

Example 11

(A) Preparation of the dialdehydostarch-deferoxamine condensationproduct as a non-radioactive carrier:

Into a solution of dialdehydostarch (10 mg) in 0.03M phosphatebuffer-0.15M aqueous sodium chloride mixture (1.0 ml), deferoxamine (23mg) was dissolved at room temperature. After addition of triethylamine(5.2 μl), stirring was continued at 12° to 15° C. for 20 minutes toobtain a solution containing the dialdehydostarch-deferoxaminecondensation product useful as a non-radioactive carrier.

(B) Preparation of the 19-9 F(ab')₂ fragment-combineddialdehydostarch-deferoxamine condensation product (a 19-9 F(ab')₂fragment-combined non-radioactive carrier):

The non-radioactive carrier (0.42 ml) as obtained in (A) was added to aphysiological saline solution of 19-9 F(ab')₂ fragment (i.e. F(ab')₂fragment of monoclonal anti-human colorectal carcinoma antibody 19-9;concentration, 18 mg/ml) (0.55 ml), followed by stirring at 4° to 6° C.for about 2 hours. After addition of sodium borohydride (3 mg), stirringwas continued at 4° to 6° C. for about 1 hour. The reaction mixture wasdialyzed to 0.05M phosphate buffer-0.15M aqueous sodium chloride mixture(pH, 5.5) at 4° to 6° C. for 24 hours and then passed through a columnof Sephadex G-150 Superfine (diameter, 2.2 cm; height, 30 cm) using0.05M phosphate buffer-0.15M aqueous sodium chloride mixture as aneluting solvent. The resultant solution was diluted with the samesolution as the eluting solvent to make a 19-9 F(ab')₂ fragmentconcentration of 0.5 mg/ml, and sodium ascorbate was added thereto tomake a concentration of 100 mM, whereby a 19-9 F(ab')₂ fragment-combinednon-radioactive carrier was obtained as a pale yellow transparentsolution.

(C) Preparation of the ⁶⁷ Ga-labeled, 19-9 F(ab')₂ fragment-combineddialdehydostarch-deferoxamine condensation product as a radioactivediagnostic agent:

To the 19-9 F (ab')₂ fragment-combined non-radioactive carrer (1 ml) asobtained in (A), a solution (0.5 ml) containing ⁶⁷ Ga (0.5 mCi) in theform of gallium citrate was added to obtain the ⁶⁷ Ga-labeled, 19-9F(ab')₂ fragment-combined dialdehydostarch-deferoxamine condensationproduct as a pale yellow transparent solution, which is useful as aradioactive diagnostic agent.

(D) Properties of the radioactive diagnostic agent as obtained in (C):

The radioactive diagnostic agent as obtained in (C) was subjected toelectrophoresis (1 mA/cm, 30 minutes) using Veronal buffer (pH, 8.6) asa developing solvent and a cellulose acetate membrane as anelectrophoretic membrane, and scanning was carried out with aradiochromato-scanner. Radioactivity was recognized as a single peak atthe locus of 1.1 cm distant from the original line towards the negativeside. This locus was the same as the coloring band of the 19-9 F (ab')₂fragment with Ponceau 3R.

From the above result, it is understood that the radioactive diagnosticagent has a labeling rate of nearly 100% and its electrostatic state issubstantially equal to that of the 19-9 F (ab')₂ fragment.

What is claimed is:
 1. A non-radioactive carrier which comprises (1) aunit of a non-physiologically active polyformyl compound having 3 to4,000 formyl groups per molecule, and (2) at least two units of an aminogroup-containing chelating compound bonded to the polyformyl compoundwith intervention of a methyleneimine linkage (--CH═N--) or amethyleneamine linkage (--CH₂ NH--) formed by a condensation between aformyl group in the polyformyl compound and an amino group in theshelating compound.
 2. The non-radioactive carrier according to claim 1,wherein the polyformyl compound is polyacrolein.
 3. The non-radioactivecarrier according to claim 2, wherein the polyacrolein comprises 10 to500 units of acrolein.
 4. The non-radioactive carrier according to claim1, wherein the polyformyl compound is a poly(dialdehydosaccharide). 5.The non-radioactive carrier according to claim 4, wherein thepoly(dialdehydosaccharide) is dialdehydostarch.
 6. The non-radioactivecarrier according to claim 4, wherein the poly(dialdehydosaccharide) isdialdehydodextran.
 7. A process for preparing a non-radioactive carrierwhich comprises condensing a non-physiologically active polyformylcompound having 3 to 4,000 formyl groups per molecule with an aminogroup-containing chelating compound to form an iminomethylene linkagebetween a formyl group in the polyformyl compound and an amino group inthe chelating compound.
 8. A chemical product which comprises(1) a unitof a non-physiologically active polyformyl compound having 3 to 4,000formyl groups per molecule, and (2) at least two units of an aminogroup-containing chelating compound bonded to the polyformyl compoundwith intervention of a methyleneimine linkage (--CH═N--) or amethyleneamine linkage (--CH₂ NH--) formed by a condensation between aformyl group in the polyformyl compound and an amino group in thechelating compound.
 9. The non-radioactive carrier according to claim 4,wherein the poly(aldehydosaccharide) is dialdehydoamylose.
 10. Thechemical product according to claim 8, wherein the polyformyl compoundis polymethacrolein having 10 to 500 repeating units of methacrolein permolecule.
 11. The non-radioactive carrier according to claim 1, whereinthe polyformyl compound is dialdehydostarch and the chelating compoundis deferoxamine.
 12. The non-radioactive carrier according to claim 1,wherein the polyformyl compound is a poly(dialdehydosaccharide) and thechelating compound is deferoxamine.
 13. A non-radioactive carrier whichcomprises (1) a unit of a non-physiologically active polyformyl compoundhaving 10 to 500 formyl groups per molecule, and (2) at least two unitsof an amino group-containing chelating compound bonded to the polyformylcompound with intervention of a methyleneimine linkage (--CH═N--) or amethyleneamine linkage (--CH₂ NH--) formed by a condensation between aformyl group in the polyformyl compound and an amino group in thechelating compound.